Method and composition for remediating environmental contaminants

ABSTRACT

A method and composition for the remediation of environmental contaminants in soil, sediment, aquifer material or water wherein contaminants are first reduced with a reducing agent found in sediment and are then oxidized to environmentally safe products. The composition includes a reducing agent, solubilized from sediment by a solvent, for reduction of environmental contaminants such as nitroorganics, halogenated hydrocarbons, cyano compounds, anisoles and metals.

This is a division of application Ser. No. 08/632,884 filed Apr. 16,1996, now U.S. Pat. No. 5,711,020, which is a continuation ofapplication Ser. No. 08/447,483, filed May 23, 1995, now abandoned,which is a division of application Ser. No. 08/045,966 filed Apr. 9,1993, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to the field of ecology and moreparticularly to the remediation of environmental contaminants byenzymatic reduction and oxidation.

Contamination of the air, water and soil is a severe problem endangeringthe lives of many plants and animals, including humans. Many attemptshave been made to reduce contamination by either preventing escape ofthe contaminants into the environment, containing the contaminants atone site, or treating the contaminants in some way to make them lessharmful.

Extensive soil, water, sediment and aquifer contamination has occurredfrom the manufacture and widespread use of explosives by both civiliansand the military. For example, the compound 2,4,6-trinitrotoluene (TNT)is a highly oxidized nitroaromatic that is stable on soil surfaces inthe environment for as many as 40 years.

Currently, the preferred technology for remediating TNT-contaminatedsoil is by burning the soil and then cashing the incinerated soil in anenclosure for an indefinite amount of time. The burning treatment isexpensive, costing $300-400 per ton, and, for a large contaminated siteencompassing several acres, could cost tens of millions of dollars.

Scientists have shown that it is possible to degrade certain organicpollutants in soil through oxidation/reduction (redox) reactions. Thegoal has been to chemically or biologically oxidize or reduce thecontaminants or functional moieties of the contaminants to innocuouscompounds or compounds that can be easily degraded and eliminated fromthe soil by known processes.

Nitroorganic Pesticides

Environmental contaminants that have been partially degraded throughredox reactions include nitroaromatic pesticides such as parathion,methyl parathion, trifluralin, profluralin, benefin, nitrofen andpentachloro-nitrobenzene as described by Williams, P. P., Residue Rev.66:63-135 (1977); Wahid, P. A. et al., J. Environ. Qual. 9:127-130(1980); Graetz, D. A. et al., J. Water Poll. Control Fed. 42:R76-R94(1970); Adhya, T. K. et al., J. Agric. Food Chem. 29:90-93 (1981);Camper, N. D. et al., J. Environ. Sci. Health B15:457-473 (1980); Golab,T. et al., J. Agric. Food Chem. 27:163-179 (1979); Probst, G. W. et al.,J. Agric. Food Chem. 15:592-599 (1967); Willis, G. H. et al., J.Environ. Qual. 3:262-265 (1974); Golab. T. et al., J. Agric. Food Chem.18:838-844 (1970); Lee, J. K. et al., Misaengmul Hakhoe Chi. 20:53-66(1982); Qian, W. W. et al., Huanjing Kexue 3:36-39 (1982); and Murthy,N. B. K. and D. D. Kaufman, J. Agric. Food Chem. 26:1151-1156 (1978).

These nitroaromatic pesticide treatments generally involve the anaerobictransformation of the pesticides to reduced compounds by unidentifiedsubstances naturally present in the soil. However, the reduced productsof nitroaromatic pesticide degradation include anilines and othercompounds that are considered to be environmental hazards.

Nitroorganic Explosives

Many unsuccessful attempts have been made to oxidize the explosives2,4,6, -trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine(RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX) andN-methyl-N-2,4,6-tetranitroaniline (Tetryl), nitrocellulose and redwater (a by-product of TNT production) to innocuous products. None havefound any practical or commercial application because these compoundsare highly oxidized, and further oxidation generally requires excessiveamounts of energy. Attempts have also been made to remediate thesecompounds microbially by reduction under anaerobic conditions. (Alvarez,M. et al., Enzyme Catalyed Transformation of 2,4,6-Trinitrotoluene,Abstracts of the General Meeting of the Am. Soc. for Microbiology 91:217(1991)) However, the reduction products of these compounds include thecorresponding amines and several other less well defined hydroxyazocompounds. The analogs of these reduction products are potentialcarcinogens and are considered to be environmentally hazardous.

Halogenated Hydrocarbons

Halogenated hydrocarbons as a class of compounds are one of the mostubiquitous pollutants in the United States. They have been and still arewidely used in many industries as cleaning solvents, refrigerants,fumigants and starting materials for the syntheses of other chemicals.Because of their extensive use, there are hundreds of contaminatedgroundwater and landfill sites in the United States, many of which aresuperfund sites for which there is no inexpensive, effective remediationtechnology available. Also, industrial waste treatment technology isexpensive and not always effective.

In contaminated ground water systems, the water is pumped out of thereservoir and treated with the "air stripping" treatment procedure.Halogenated hydrocarbons have also been remediated by a photolysisprocedure wherein contaminated soil or sediment is placed on an oxidefilm and irradiated with concentrated sunlight to remove chloride atoms.These procedures are expensive and only successful if all of thecontaminated material has been successfully removed from the site ofcontamination. Effective in situ treatment is not practiced because of alack of treatment technology. Bioremediation has not been successfulbecause maintenance of a viable microorganism population is notgenerally feasible in subsurface ecosystems. Chemical remediationprocesses have not been utilized because of the delivery of largeamounts of the necessary chemicals and problems associated withgroundwater hydrology.

Bioremediation has received considerable attention as an in situremediation process of contaminated waste sites. The parent pollutants,however, are often resistant to degradation and must first betransformed to more degradable compounds for the processes to beeffective. Although many microorganisms have been isolated that arecapable of degrading halogenated hydrocarbons in the laboratory, theyare not always effective when ported to the field situation.

Cyano Compounds

Aliphatic and aromatic cyano compounds are used as solvents andintermediates in the chemical industry in a variety of syntheticprocesses including textiles and pesticides. For example, acrylonitrileis a high production compound with output exceeding more than 2.3billion pounds a year. These compounds can enter the environment throughmanufacturing waste waters and from the polymers of which they areassociated and as a result of applications of pesticides such asdichlobneil (2,6-dichlorobenzonitrile) and bromoxynil(3,5-dibromo-4-hydroxybenzonitrile).

Biodegradation of selected cyano compounds has been demonstrated inwaste water treatment systems. In soils, however, degradation is moredifficult and high concentrations of the pollutants are often notreadily degraded. Also, not all soils, in particular sandy soils, havethe necessary microbial populations to degrade nitrites.

Anisoles

Anisoles are used as intermediates in the chemical industry for themanufacture of a large number of polymer, dye and pesticide compounds.These compounds find their way into the environment through point sourceand non-point source pathways. For example, the pesticide methoxyclorhas been one of the most widely used pesticides in the United States. Ingeneral, anisoles are hard to degrade because the methyl-oxygen bond isvery strong.

Other Contaminants

Remediation of other environmental contaminants such as metals has alsobeen largely unsuccessful. Metal contaminants have been treated by the"pump and treat" procedure wherein water is pumped out of thecontaminated area and passed over a tube containing titanium totransform the contaminants to compounds that are less hazardous to theenvironment. The "pump and treat" method is very costly, is onlyapplicable for removal of volatile contaminates from surface water oraquifers, and is not successful until the source of the contamination isdepleted.

It would be of great environmental benefit to have an inexpensive methodof degrading contaminants in soils, waters, sediments, and aquifermaterials that results in products that are environmentally acceptable.

It is therefore an object of the present invention to provide animproved method of remediating environmental contaminants.

It is a further object of the present invention to provide a method ofremediating environmental contaminants that can be carried out in situand in batch reactors.

It is a further object of the present invention to provide a compositionfor rapid reduction of contaminants.

It is a further object of the present invention to provide a process forthe production of contaminant-reducing agents from soil.

It is a further object of the present invention to provide a method ofremediating nitro-, halogenated-, cyano-, methoxy-organic, and metalcontaminants from the environment.

It is a further object of the present invention to provide a method ofoxidizing reduced pollutants.

It is a further object of the present invention to provide a method ofremediating environmental contaminants that is cost-effective.

It is a further object of the present invention to provide a method ofremediating soils, sediments, and aquifers that maintains the integrityof the environmental compartment.

SUMMARY OF THE INVENTION

A method for the remediation of contaminants of soil, water, sedimentand aquifers is disclosed wherein contaminants are first reduced by areducing agent found in soil and sediment having a substantially highorganic content and are then oxidized to environmentally safe products.Reduction preferably takes place in a substantially anaerobicenvironment. An anaerobic environment is naturally present in water andaquifers and can be created in contaminated soil or sediment by floodingthe soil or sediment with water.

Reduction of contaminants is achieved by combining the contaminatedsoil, water, sediment or aquifer material with either soil containing anadequate amount of the reducing agent, a crude enzyme preparationcontaining the reducing agent, the reducing agent as a semi-purified orpurified enzyme specific for reduction of a particular contaminant, or acombination thereof. The contaminated soil, water, sediment or aquifermaterial is incubated with the reducing agent for a sufficient amount oftime to allow reduction of the contaminants. The addition of a reducingmetal, such as iron, to the soil further accelerates the remediationprocess.

Oxidation is achieved by oxygenating the water, aquifer material,flooded soil, or sediment containing the reduced contaminant or bysimply removing the water from flooded soil or sediment containing thereduced contaminant.

A method for preparing the crude soil enzyme extract or isolated enzymespecific for reduction of specific environmental contaminants isdisclosed wherein the reducing agent is extracted from soil by combiningthe soil with a solvent that solubilizes proteins. Preferably theextract is prepared from soil having a relatively high carbon content sothat it contains a higher concentration of reducing agents. Furtherpurification of the reducing agent can be achieved by proteinprecipitation and fractionation on chromatography columns. Semi-purifiedenzymes particularly useful for reducing specific classes ofenvironmental contaminants and the processes for isolating theseproteins are provided herein.

Contaminants that can b e reduced by the remediation method describedherein include nitroorganics in general and specifically munitions suchas TNT, RDX, HMX, nitrocellulose, and red water and pesticides such asmethyl parathion and 2-(sec-butyl)-4,6-dinitrophenol, also known asDinoseb; halogenated organic compounds such as halogenated organicsolvents, halogenated pesticides and other industrial halogenatedcompounds such as and pentachlorophenol; cyano compounds such asbenzonitrile, acetonitrile, and other industrial chemicals; anisolessuch as anisole, dyes and pesticides containing methoxy moieties; andmetals such as chromium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the relative reducing activity of a fractionatedcrude enzyme sediment extract. Fractionation was performed on a QAECellulose ion exchange chromatography column. The symbol ▪ representsthe concentration of protein as μg protein per 50 μl aliquot of eachfraction. The symbol  represents relative TNT-reducing activity. Thesymbol ▴ represents relative 4-chlorobenzonitrile-reducing activity.

FIG. 2 is a graph of relative reducing activity of the void volumefractions of FIG. 1 after fractionation on a SEPHAROSE™ CL-6B sizeexclusion chromatography column. The symbol ▪ represents theconcentration of protein as μg protein per 50 μl aliquot of eachfraction. The symbol  represents relative TNT-reducing activity. Thesymbol ▴ represents 4-chlorobenzonitrile-reducing activity. The opensquare symbol represents relative PCE-reducing activity.

FIG. 3 is a graph of relative reducing activity of fraction 18 of FIG. 2after fractionation on a Phenyl SEPHAROSE™ hydrophobic interactionchromatography column. The symbol ▪ represents the concentration ofprotein as μg protein per 50 μl aliquot of each fraction. The symbol represents relative TNT-reducing activity.

FIG. 4 is a graph of relative reducing activity of fraction 18 of FIG. 2after fractionation on a Zn:Iminodiacetic Acid SEPHAROSE™ 6Bchromatography column. The symbol ▪ represents the concentration ofprotein as μg protein per 50 μl aliquot of each fraction. The symbol represents relative TNT-reducing activity. The symbol ▴ represents4-chlorobenzonitrile-reducing activity.

FIG. 5 is a bar graph of percent TNT-reducing activity inhibition afteraddition of protease to fraction 18 of FIG. 2.

FIG. 6 is a graph of relative reducing activity for fractions 4, 13, and19 from FIG. 2 after exposure to various temperatures. The symbol represents fraction 4. The symbol ▴ represents fraction 13. The symbol ▪represents fraction 19.

FIG. 7 shows chemical structures for the nitroorganics Disperse Blue 79,Disperse Red 5, parathion, dinoseb, RDX, HMX, and TNT.

FIG. 8 is a schematic representation of the stepwise reduction ofsubstituted nitrobenzenes to the corresponding anilines in anaerobicsediments.

FIG. 9 is a graph of the reduction of nitrobenzene to nitrosobenzene,phenylhydroxyamine and aniline after incubation under anaerobicconditions with a sediment sample containing a nitrobenzene reducingagent. The black triangle symbol represents nitrobenzene, the blacksquare symbol represents phenylhydroxyamine, the black circle symbolrepresents nitrosobenzene, the open square symbol represents aniline,and the asterisk symbol represents the sum of components.

FIG. 10 is a schematic representation of the stepwise oxidation ofanilines to catechols, carbon dioxide and the corresponding acetates.

FIG. 11 is a graph showing the relationship between the disappearancerate constant of nitrobenzene and the organic carbon content of sedimentwhen nitrobenzene is incubated under anaerobic conditions with varioussediment samples containing a nitrobenzene reducing agent. (R² =0.968;X1=0.442; C=-2.54)

FIG. 12 is a representative HPLC chromatogram showing the relevantretention times and separation of TNT from the reduction products of TNTafter incubation with a TNT reducing agent.

FIG. 13 is a graph showing the rate of reduction of variousconcentrations of TNT in an anaerobic sediment sample. The symbol ▪represents an initial TNT concentration of 125 ppm. The symbol +represents an initial TNT concentration of 2.5 ppm. The open diamondsymbol represents an initial TNT concentration of 0.25 ppm.(p=0.12±0.01; pH=6.5; Eh=-368 mv (Ag/AgCl))

FIG. 14 is graph comparing TNT reduction kinetics in aquifer materialafter incubation with a protein extract containing a TNT reducing agent.The x-containing open square symbol represents TNT in aquifer materialalone. The t_(1/2) is 70.6 hours. The x-containing open diamond symbolrepresents TNT plus protein extract. The t_(1/2) is 6.5 hours. Thex-containing open triangle symbol represents TNT in aquifer materialplus protein extract. The t_(1/2) is 4.2 hours.

FIG. 15 is a graph showing the reduction of 1.6 ppm TNT in an aqueoussolution of 1% iron (w/v) in the absence of reducing agent. The t_(1/2)is 1.14 days and r² =0.99.

FIG. 16 is a graph showing TNT reduction in flooded contaminated soilsamples. The first arrow indicates that iron was added after 48 days,and the second arrow indicates that the sample was inverted to mix thecontents after 51 days. The solid square represents supernatant. Theopen triangle represents sediment. The open square represents Eh.

FIG. 17 is a graph showing the enzymatic reaction of trichloroethane(TCE) as concentration divided by initial concentration versus time. Theinitial concentration was 97.0 μM. The black circle symbol representsthe control. The dash symbol represents TCE.

FIG. 18 is a graph showing reactivity (percent activity) oftetrabromoethene (PBE) and hexachloroethane (HCA) with protein as afunction of temperature. The black circle symbol represents HCA. Theblack square symbol represents PBE.

FIG. 19 is a graph showing reactivity (percent activity) oftetrabromoethene (PBE) and hexachloroethane (HCA) with protein as afunction of pH. The black circle symbol represents HCA. The black squaresymbol represents PBE.

FIG. 20 is a graph showing a decrease in the concentration ofhexachloroethane (HCA) after reaction with the dehalogenase enzyme.

FIG. 21 is a graph showing the Michaelis-Menton Kinetics fortetrabromoethene (PBE).

DETAILED DESCRIPTION OF THE INVENTION

A method and composition for remediating a contaminant in soil, water,sediment, and aquifers to an environmentally safe product are provided.The environmental contaminant is first reduced by reacting thecontaminant with a reducing agent. The reducing agent is an enzyme orcombination of enzymes found in soil or sediment and is generally foundin greater concentrations in soil having a higher organic content. Thecontaminant is reacted with the reducing agent by combining thecontaminant with soil, a crude soil extract containing an amount of thereducing enzymes, or semi-purified or purified reducing enzymes specificfor the reduction of a selected class of compounds sufficient to causereduction. The reduced contaminant is then oxidized to anenvironmentally innocuous product by exposure to oxygen or air. Themethod is applicable to both in situ and batch processing and can beused to remediate contaminants such as nitroorganics, halogenatedhydrocarbons, anisoles, cyano compounds, and metals as discussed in moredetail below.

Reduction of Soil or Sediment Contaminants

In the method described herein, contaminated soil or sediment is firstdeprived of oxygen to create a substantially anoxic or anaerobicenvironment. While an anoxic environment is not essential for reductionof the contaminants by the reducing enzymes, it facilitates reduction bymaking the environment unfavorable to aerobic organisms. It has beendiscovered that aerobic organisms produce proteases that cleave andthereby inactivate the reducing enzymes used in the remediation methodprovided herein. In a preferred embodiment, the contaminated soil orsediment is deprived of oxygen by flooding the surface area of the soilor sediment with water. Contaminated sediment need not be flooded withwater if it already exists in an anoxic state. The contaminated soil orsediment is then incubated for a sufficient period of time under theanoxic conditions to allow reduction of the contaminants by reducingagents either naturally present in the contaminated soil or sediment orby reducing agents added to the contaminated soil or sediment in theform of a crude enzyme extract or semi-purified enzyme extracted fromsoil. Mass transfer limitations can be overcome by mechanical mixing.

The term "reducing agents" as used herein refers to substances whichfacilitate the reduction of a compound. If the contaminated soil orsediment has a relatively low carbon content or if the degree ofcontamination is extensive and the soil therefore tails to contain anadequate concentration of reducing agents for rapid or completereduction of the contaminants, a crude soil extract containing anadequate concentration of reducing enzymes for reduction of thecontaminants, a semi-purified or purified enzyme or enzymes specific forreduction of the particular contaminant to be remediated, or acombination thereof may be added to the contaminated soil or sedimentfor more rapid and complete contaminant reduction. Soil or sedimenthaving a "substantially high organic or carbon content" as used hereingenerally excludes soils containing predominantly sand or clay. Forexample, sediment collected from the Beaver Dam stream, near Athens,Ga., having a organic carbon content of 3% contains approximately 100 μgprotein per liter of sediment. The amount of crude or purified enzymeadded depends on the compound being remediated and the activity of theenzyme. For example, 1 μg of protein reduces 10⁻⁶ M TNT in approximately15 minutes.

The extent of incubation depends on the carbon content of the soil orsediment which directly relates to the concentration of reducing enzymesin the soil and the amount of contamination as described above. Ingeneral, the higher the organic carbon content, the faster thecontaminants are reduced. Preferably, the soil or sediment is incubatedfrom one to four days. The addition of iron, preferably in the form ofiron powder or filings provides an additional source of electrons, and,when added to the incubation mixture, greatly accelerates the reductionreaction.

Preferably, the soil or sediment being remediated is maintained in thepH range of 5 to 8 for both the oxidation and reduction steps. Theheterogenous soil and water phase provides the pH buffering capacity.The Eh of the system is preferably -50 mV or less (relative to Ag/AgCl).In addition, the temperature of the contaminated area during remediationpreferably ranges from 10 to 115° C. with the optimal temperatureranging between 25 and 37° C.

The sediment or flooded soil preferably contains a soil to water ratioranging from 0.02:1 to 0.5:1 (g:g). It has been discovered that, inbatch processes, the rate constants for reduction increase withincreasingly greater soil to water ratios up to a ratio of 0.5:1.

Reduction of Water or Aquifer Material Contaminants

In the method described herein for remediation of contaminated water oraquifers, a sufficient amount of soil having a substantially highorganic carbon content, a crude soil extract containing reducingenzymes, or a semi-purified or purified enzyme specific for reduction ofthe contaminant is combined with the contaminated water or aquifermaterial for reduction of the contaminants. As described above, theenzyme-containing soil and the water can be mixed by mechanical means toovercome mass transfer limitations. Contaminated water and aquifers arenaturally anoxic and need no removal of oxygen during the incubationstep.

The pH and Eh values, soil to water ratio, and extent of incubation arepreferably the same as described above for the reduction of soil andsediment contaminants. As described above, the addition of iron to theincubation mixture, preferably in the form of iron powder or filings,greatly accelerates the reduction reaction.

Isolation of Reducing Agents

a) Extraction

The crude enzyme extract and semi-purified or purified enzyme describedabove for reduction of soil, sediment, water, or aquifer materialcontaminants are extracted from a soil sample with one or more of avariety of different solvents. Preferably, the soil sample has asubstantially high organic carbon content and therefore contains aconcentrated amount of the reducing agent or agents as described above.Most preferably, the soil sample is a sediment rich in organic materialsuch as the type of sediment found in a marsh or bog.

The source of the enzyme is believed to be a common aquatic plant, orweed, such as the hornwort moss. Therefore, the soil from which theenzyme is extracted should contain hornwort moss or should havepreviously supported the growth of the hornwort moss. The hornwort moss,also known as the horned liverwort, is a mosslike lower plant having noflowers. The hornwort moss is a creeping annual or perennial plant ofthe class Anthocerotopsida. In some classification systems, hornwortsare grouped as horned liverworts in the subclass Anthocerotidae (classHepaticae), class Anthocerotopsida, order Anthocerotales, or areclassified in the division Anthocerotophyta. Hornworts usually grow ondamp soils or on rocks in tropical and warm temperate regions. Rhizoids(rootlike structures) on the undersurface anchor the plant.

Optimal enzymatic yield is obtained when the soil sample is removed frombeneath a water surface and is stored and transported undersubstantially anoxic conditions to limit proteolytic cleavage by aerobicorganisms naturally present in the sample.

The crude enzyme extract is prepared from the soil sample by gentlymixing approximately one volume of the sample with one or more volumesof solvent and then separating the solubilized proteins from the soil bymechanical methods known to those skilled in the art, such ascentrifugation. Any solvent or mixture of solvents in which proteins aresolubilized may be used to solubilize the reducing agent. A list ofsolvents useful for solubilizing the reducing reagent is set forth belowin Table 1. Preferred solvents contain glycerol or methanol. The mostpreferred solvent is 20% glycerol in either water or a buffer, such asTris or phosphate at pH 8. High quality solvents such as commercial highpressure liquid chromatography (HPLC) grade solvents are preferred tominimize additional contamination. HPLC grade solvents can be obtainedfrom commercial chemical suppliers such as Burdick and Jackson, Musegon,Mich. The solvents sodium dodecyl sulfate (SDS), CTMA BR(cetyltrimethylammonium bromide), Triton X-100 and Tween 20 aredetergents, whereas ammonium sulfate, urea, potassium phosphate andammonium phosphate are salts.

                  TABLE 1                                                         ______________________________________                                        Solvents Useful for Extracting                                                  Reducing Reagent                                                            ______________________________________                                        0.5% sodium dodecyl sulfate (SDS) in water                                      0.5% cetyltrimethylammonium bromide (CTMA Br) in                              water                                                                         0.5% Triton X-100 in water                                                    0.5% Tween 20 in water                                                        20% glycerol in water                                                         20% glycerol + 0.5% SDS in water                                              20% glycerol + 0.5% CTMA Br in water                                          20% glycerol + 0.5% Triton X-100 in water                                     20% glycerol + 0.5% Tween 20 in water                                         500 mM KCl                                                                    500 mM KCl + 0.5% SDS in water                                                500 mM KCl + 0.5% CTMA Br in water                                            500 mM KCl + 0.5% Triton X-100 in water                                       500 mM KCl + 0.5% Tween 20 in water                                           2 M NH.sub.4 SO.sub.4                                                         2 M NH.sub.4 SO.sub.4  + 0.5% SDS in water                                    2 M NH.sub.4 SO.sub.4  + 0.5% CTMA Br in water                                2 M NH.sub.4 SO.sub.4  + 0.5% Triton X-100 in water                           2 M NH.sub.4 SO.sub.4  + 0.5% Tween 20 in water                               6 M urea                                                                      6 M urea + 0.5% SDS in water                                                  6 M urea + 0.5% CTMA Br in water                                              6 M urea + 0.5% Triton X-100 in water                                         6 M urea + 0.5% Tween 20 in water                                             K.sub.2 HPO.sub.4  + KH.sub.2 PO.sub.4  pH 7 (0.5 M)                          K.sub.2 HPO.sub.4  + KH.sub.2 PO.sub.4  pH 7 (0.5 M) + 0.5% SDS in          water                                                                           K.sub.2 HPO.sub.4  + KH.sub.2 PO.sub.4  pH 7 (0.5 M) + 0.5% CTMA Br in      water                                                                           K.sub.2 HPO.sub.4  + KH.sub.2 PO.sub.4  pH 7 (0.5 M) + 0.5% Triton          X-100 in water                                                                  K.sub.2 HPO.sub.4  + KH.sub.2 PO.sub.4  pH 7 (0.5 M) + 0.5% Tween 20 in     water                                                                          0.5 M NH.sub.4 HPO.sub.4                                                      0.5 M NH.sub.4 HPO.sub.4  + 0.5% SDS in water                                 0.5 M NH.sub.4 HPO.sub.4  + 0.5% CTMA Br in water                             0.5 M NH.sub.4 HPO.sub.4  + 0.5% Triton X-100 in water                        0.5 M NH.sub.4 HPO.sub.4  + 0.5% Tween 20 in water                            20% glycerol + 50 mm Tris-HCl, pH 8.0 in water                               ______________________________________                                    

Most preferably, the sample and solvent mixture is allowed to stand forseveral minutes until the majority of the particular matter of thesample has precipitated and the proteins have been solubilized. Thesupernatant is decanted and centrifuged for approximately 20 minutes at5000 rpm to remove the finer particulate matter. The extraction andcentrifugation steps can be repeated to increase yield. The resultingsupernatant is a crude enzyme extract that can be used to reduceenvironmental contaminants without further purification if desired.

b) Protein Precipitation

The crude enzyme extract can be concentrated and further purified byprecipitating the proteins from the extract. Salt precipitation with aconcentration sufficient to achieve 80-90% saturation with a salt suchas ammonium sulfate is preferred.

c) Desalting

A semi-purified enzyme extract can be prepared by resuspending theprecipitate in water and simultaneously removing the salt andfractionating the proteins on a desalting or size exclusion column suchas a SEPHADEX™ G-25sf column, available from Pharmacia, Inc.,Piscataway, N.J. The SEPHADEX™ G-25sf column fractionates moleculeshaving a molecular weight greater than 5000, therefore the fractioneluting in the void volume of the G-25 column contains molecules havinga molecular weight greater than 5000. This semi-purified high molecularweight enzyme fraction contains most of the reducing activity for allclasses of contaminants remediated by the method described herein exceptmetals. Therefore, the enzymes useful for remediation of nitroorganics,halogenated hydrocarbons, anisoles, and cyano compound contaminants allhave a molecular weight greater than 5000. Metal contaminants, such asK₂ CrO₄, are reduced by the low molecular weight fraction from the G-25column (less than 5000 daltons) or the total extract. Therefore, theenzyme or compounds specific for reduction of metal contaminants have amolecular weight less than 5000 daltons.

d) Additional Purification

The enzyme extract can be further purified using conventional proteinpurification techniques known to those skilled in the art, alone or incombination, for the isolation of substantially pure enzyme. Proteinpurification techniques include separation of the active enzyme bycharge fractionation using an ion exchange column such as an QAE anionexchange or DEAE SEPHAROSE™ anion exchange column (Pharmacia,Piscataway, N.J.); by size fractionation using a size exclusion columnsuch as a CL-6B SEPHAROSE™ column (Pharmacia); by hydrophobicityfractionation using a phenyl column such as a phenyl SEPHAROSE™ CL-4Bcolumn (Pharmacia); and by amino acid metal binding properties using azinc affinity column such as a Zinc Iminodiacetic Acid-SEPHAROSE™ 6BFast Flow Column (Sigma Chemical Co., St. Louis, Mo.).

Enzymes specific for the reduction of nitroorganics, halogenatedhydrocarbons, anisoles, cyano compounds and metals can be isolated fromthe crude enzyme extract using a combination of the above-mentionedprotein fractionation techniques as described in more detail below.Fractions are assayed for activity as described below, and activefractions are pooled prior to fractionation by a subsequent separationmechanism.

e) Assay for Reducing Activity

Fractions collected during each step of the enzyme purification methoddescribed herein can be assayed for reducing activity by combining analiquot of each fraction with a solution containing a compound from theclass of contaminants to be remediated. The mixture is incubated andassayed for the presence of reduced contaminants by analytical methodsknown to those skilled in the art, such as HPLC and GC.

Protein concentrations for each fraction can be determined usingcommercially available reagents such as the BIORAD™ Protein Reagent(BioRad, Richmond, Calif.) in accordance with the manufacturer'sinstructions or by conventional protein analysis methods well known tothose skilled in the art.

f) Isolation of Reducing Agent with Monoclonal Antibodies

The reducing agent, or a particular enzyme specific for the reduction ofa particular class of contaminants such as a nitroreductase, nitrilase,demethylase, or dehalogenase can be isolated from a soil sample, or asoil sample can be tested for the presence of such an enzyme, with theaid of a detectable polyclonal or monoclonal antibody in accordance withmethods well known to those skilled in the art. The production ofmonoclonal antibodies specific for a particular enzyme or protein isalso achieved by methods well known to those skilled in the art, such asthe methods described in Antibodies, A Laboratory Manual, Ed Harlow,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1988.

Basically, monoclonal antibodies are produced from a hybridoma cellgenerated by fusing a normal antibody-producing lymphocyte from thespleen of an experimental animal, such as a mouse or rat, recentlyimmunized with the protein of interest, to a myeloma cell line that doesnot synthesize its own immunoglobulin and is deficient in the enzymehypoxanthine-guanine phophoribosyltransferase (HGPRT), which catalyzesreactions of the bases hypoxanthine and guanine with5-phophoribosyl-1-pyrophosphate to form the nucleotidesinosine-5'-P(IMP) and guanosine-5'-P (GMP), respectively. The hybridcells are selected over either parental cell by culturing in a mediumcontaining hypoxanthine, aminopterin, and thymidine (HAT medium). Themyeloma cells, lacking HGPRT, cannot survive because the de novosynthesis of GMP is blocked by the folate antagonist aminopterin, andthe normal lymphocytes grow very slowly in HAT medium, whereas thehybrid cells, in which a functional HGPRT gene is supplied by thelymphocyte genome, grow rapidly and form large colonies that are readilydistinguishable from those of the slowly growing lymphocytes. Clones ofhybrid cells producing the desired antibody are identified by a suitableassay procedure and grown into larger cultures. The homogenousimmunoglobulin produced by such a cloned hybrid is a monoclonal antibodyhaving specificity for the enzyme.

The monoclonal antibody can be made detectable by attachment of adetectable label. The various types of labels and methods of labellingmonoclonal antibodies are well known to those skilled in the art.Several specific labels are set forth below.

For example, the label can be a radiolabel such as, but not restrictedto, ³² P, ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I, or ¹³¹ I. Detection of a radiolabelcan be by methods such as scintillation counting, gamma ray spectrometryor autoradiography.

The label can also be a Mass or Nuclear Magnetic Resonance (NMR) labelsuch as, for example, ¹³ C, ¹⁵ N, or ¹⁹ O. Detection of such a label canbe by Mass Spectrometry or NMR.

Fluorogens can also be used to label the monoclonal antibodies. Examplesof fluorogens include fluorescein and derivatives, phycoerythrin,allo-phycocyanin, phycocyanin, rhodamine, Texas Red or other proprietaryfluorogens. The fluorogens are generally attached by chemicalmodification. The fluorogens can be detected by a fluorescence detector.

The monoclonal antibodies can alternatively be labelled with a chromogento provide an enzyme or affinity label. For example, the antibody can bebiotinylated so that it can be utilized in a biotin-avidin reactionwhich may also be coupled to a label such as an enzyme or fluorogen. Theprobe can be labelled with peroxidase, alkaline phosphatase or otherenzymes giving a chromogenic or fluorogenic reaction upon addition ofsubstrate. For example, additives such as5-amino-2,3-dihydro-1,4-phthalazinedione (also known as LUMINOL™) (SigmaChemical Company, St. Louis, Mo.) and rate enhancers such asp-hydroxybiphenyl (also known as p-phenylphenol) (Sigma ChemicalCompany, St. Louis, Mo.) can be used to amplify enzymes such ashorseradish peroxidase through a luminescent reaction; and luminogeneicor fluorogenic dioxetane derivatives of enzyme substrates can also beused.

A label can also be made by detecting any bound antibody complex byvarious means including immunofluorescence or immuno-enzymaticreactions. Such labels can be detected using enzyme-linked immunoassays(ELISA) or by detecting a color change with the aid of aspectrophotometer.

Reducing Agent Characteristics

The active reducing agent consists of one or more enzymes believed to bederived from the hornwart moss. The enzyme is soluble in water and maybe a membrane-bound enzyme complex or portion thereof, possibly aglyco-protein. It is believed that the enzyme is an electron carrierthat transfers electrons to the contaminants in the presence of anelectron source (anoxic conditions) and removes electrons from thecontaminants in the presence of an electron sink (oxic conditions).

The reducing agent extracted from soil or sediment as described hereinis abiotic in that neither microbial activity nor metabolism isrequired. Furthermore, with the exception of the dehalogenase enzymedescribed in more detail below, which is oxygen sensitive, the reducingagent does not require strict anaerobic conditions for activity.Treatment of the reducing agent with protease K, a digestive enzyme thathydrolyses peptide bonds in polypeptide chains, decreases the ability ofthe reducing agent to reduce contaminants thereby demonstrating that theactive component of the reducing agent is a protein.

Isolation and Characterization of the Nitroorganic Reducing Agent

An enzyme specific for reduction of nitroorganic compounds was isolatedfrom the semi-purified enzyme extract described above by the followingprocedure.

Charge

Void volume fractions from the SEPHADEX™ G-25sf column containingproteins having a molecular weight greater than 5000 daltons werecollected and pooled. The pooled fractions were concentrated with aZETAPREP™ 15 disk (Pharmacia, Piscataway, N.J.), adjusted to pH 8.6 witha 100 mM Tris buffer, and subjected to low pressure ion exchangefractionation by passage through a QAE or DEAE SEPHAROSE™ anion exchangecolumn. The mobile phase was 100 mM Tris buffer, pH 8.6. Under theseconditions, fractions having a neutral or positive charge at pH 8.6passed through the column into the void volume while fractions having anegative charge were retained on the column. Fractions were collectedand assayed for protein concentration and the ability to reduce thenitroorganic compounds TNT and 4-chlorobenzonitrile as described above.The highest levels of activity for reducing both TNT and4-chlorobenzonitrile were found in the fractions having a neutral orpositive charge at pH 8.6 (void volume fractions) as shown in FIG. 1.

Size

The fractions having high reducing activity were pooled, precipitated toreduce the volume, resuspended in 50 mm Tris, at pH 7, and subjected tofractionation by size exclusion chromatography by passage through aSEPHAROSE™ CL-6B column. The mobile phase was 50 mm Tris, pH 7.Fractions were once again assayed for protein content and the ability toreduce the nitroorganic compounds TNT and 4-chlorobenzonitrile. Themolecular weight range for each fraction was determined by comparing theelution time of the active fractions to the elution time of proteinsstandards having known molecular weights. Fractions containing moleculeshaving a molecular weight between 300,000 and 400,000 daltons, between49,000 and 80,000 daltons, and between 18,500 and 27,000 daltonsexhibited the highest levels of TNT reducing activity as shown in FIG.2, while fractions containing molecules having a molecular weight ofapproximately 600,000 daltons exhibited the highest levels of4-chlorobenzonitrile reducing activity and molecules having a molecularweight of approximately 400,000 to 600,000 daltons had the highestlevels of 1,2,3,4-tetrachloroethene (PCE) reducing activity.

Hydrophobicity

The fractions having the largest molecular weight molecules withTNT-reducing activity were pooled, precipitated, reconstituted in 1.7 Mammonium sulfate, pH 7 and passed through a Phenyl SEPHAROSE™ CL4Bcolumn for separation and fractionation on the basis of hydrophobicity.Molecules were eluted with a linear gradient of 1.7 M ammonium sulfateto 1.7 M ammonium phosphate, pH 7. The gradient was begun after fivefractions were collected. Under these conditions, hydrophilic componentspass through the column quickly while hydrophobic components areretained and are eluted as the concentration of ammonium sulfate in themobile phase is reduced. Fractions were assayed for protein content andthe ability to reduce TNT. The highest TNT reducing activity wasobserved in the hydrophobic fractions, with maximal reducing activitycontained in fraction 32, as shown in FIG. 3.

Metal-binding Ability

The active fractions were pooled, precipitated, reconstituted in 1 Mpotassium chloride and passed through a Zn Iminodiacetic acid-SEPHAROSE™CL-6B Fast Flow column. In this column, any material that does not bindzinc is eluted early. The sample was loaded onto the column in 1 Mpotassium chloride in 50 mM K₂ PO₄, washed with five void volumes of 50mM potassium phosphate buffer, pH 7, containing 1 M KCl and eluted witha linear gradient from 1 M potassium chloride to 2 M ammonium sulfate inphosphate buffer, pH 7. As shown in FIG. 4, fractions containingTNT-reducing activity are found in fractions 16-18 and bind zinc to someextent, whereas fractions containing 4-chlorobenzonitrile-reducingactivity are found in fractions 6-8 and bind zinc to a lesser extent. Ametals analysis of the active fractions revealed that the TNT-reducingenzyme contains iron whereas the 4-chlorobenzonitrile reducing enzymedoes not contain any metals.

Enzymatic Digestion

Treatment of an aliquot from pooled fractions 16-18 from FIG. 4, havingTNT-reducing or fractions 6-8, having 4-chlorobenzonitrile-reducingactivity, with increasingly larger concentrations of pronase E, proteaseK or subtilisin caused inhibition of TNT-reducing activity, as shown inFIG. 5. Treatment with the proteases chymotrypsin and trypsin, known tocleave proteins only at aromatic amino acids, caused no inhibition ofTNT reducing activity. Therefore, the TNT reducing enzyme has fewavailable aromatic amino acids. The activity of the nitrilase wasinhibited by both trypsin and chymotrypsin.

Temperature Effects

FIG. 6 shows the effect of elevated temperature on low, intermediate,and high molecular weight fractions eluted from the SEPHAROSE™ agaroseCL-6B size exclusion column. (The molecular weights of these fractionsare shown in FIG. 2.) Elevation of temperature had little effect on thehigh molecular weight fraction (fraction 4), moderate effect on theintermediate molecular weight fraction (fraction 13) and a greatereffect on the low molecular weight fraction (fraction 19).

Kinetics

The kinetics of the purified TNT-reducing agent and the purifiednitrile-reducing agent, isolated as pooled fractions 16-18 from theZn-Iminodiacetic acid-SEPHAROSE™ agarose CL-6B column, respectively, areset forth below in Table 2.

                  TABLE 2                                                         ______________________________________                                        Reducing Agents Kinetics                                                           Compound     (Concentration)                                                                             Half-life                                     ______________________________________                                        TNT           (1.5 × 10-6)                                                                          15 minutes                                          TNT (3.0 × 10-6) 30 minutes                                             TNT (7.5 × 10-6) >5 hours                                               nitrobenzene (1.0 × 10-6) 28 hours                                    ______________________________________                                    

Molecular Weight

The size of the TNT-reducing enzyme, as determined by polyacrylamide gelelectrophoresis, is approximately 316,000 daltons and is believed tocontain six subunits having a molecular weight of approximately 19,000,two subunits having a molecular weight of approximately 37,000, twosubunits having a molecular weight of approximately 66,000, and at leasttwo iron molecules. The TNT-reducing enzyme exhibits a dumbbell shapewhen viewed by scanning tunnelling microscopy.

The size of the 4-chlorobenzonitrile-reducing enzyme has a molecularweight of approximately 600,000 and is believed to be composed of foursubunits each having a molecular weight of approximately 150,000.

Isolation and Characterization of the Halogenated Hydrocarbon ReducingAgent

An enzyme specific for reduction of halogenated hydrocarbons, or adehalogenase, was isolated from the semi-purified enzyme extractdescribed above by the procedure described above for the isolation ofthe nitroorganic reducing agent with the exception that all the bufferswere kept anaerobic.

Sequential dehalogenation of a halogenated hydrocarbon with thedehalogenase results in the formation of the dehalogenated hydrocarbon.The final product appears to be carbon dioxide, a compound that isacceptable to the environment.

Charge

As described above, void volume fractions from the SEPHADEX™ G-25sfcolumn containing proteins having a molecular weight greater than 5000daltons were collected, pooled, concentrated and subjected to lowpressure ion exchange fractionation by passage through a QAE or DEAESepharose™ anion exchange column so that fractions having a neutral orpositive charge at pH 8.6 passed through the column into the void volumewhile fractions having a negative charge were retained on the column.Fractions were collected and assayed for protein concentration and theability to reduce the halogenated hydrocarbon tetrabromoethene (TBE) asdescribed above. The highest levels of activity for reducing TBE werefound in the fractions having a neutral or positive charge at pH 8.6(void volume fractions) as shown in FIG. 1.

Size

The fractions having high reducing activity were pooled, fractionated bysize exclusion chromatography by passage through a Sepharose™ CL-6Bcolumn as described above, and once again assayed for protein contentand the ability to reduce the halogenated hydrocarbon TBE. The molecularweight range for each fraction was determined as describe above.Fractions containing molecules having a molecular weight ofapproximately 293,000 daltons exhibited the highest levels of TBEreducing activity.

Metal-binding Ability

A metals analysis of the active fractions revealed that the dehalogenaseenzyme contains copper and iron.

Temperature and pH

The purified dehalogenase exhibited maximal activity at a temperaturebetween approximately 10 and 50° C. and at a pH between approximately 3and 7. The maximal activity for HCA dehalogenation was observed at atemperature between approximately 10 and 50° C., as shown in FIG. 18,and at a pH between approximately 3.8 and 6.0, as shown in FIG. 19,whereas the maximal activity for PBE dehalogenation was observed at atemperature between approximately 25 and 50° C., as shown in FIG. 18,and at a pH between approximately 5.0 and 7.0, as shown in FIG. 19.

Kinetics

The kinetics for the reduction of the following halogenated alkanes andalkenes: tetrabromoethene (PBE), hexachloroethane (HCA),tetrachloroethene (PCE) and trichloroethene (TCE), by the purifieddehalogenase, using microgram quantities of protein in and nearsaturated solutions of the halogenated hydrocarbons, are set forth belowin Table 3.

                  TABLE 3                                                         ______________________________________                                        Dehalogenase Reducing Agents Kinetics                                              Compound     (Concentration)                                                                             Half-life                                     ______________________________________                                        PBE           (30 μM)    1100 minutes                                        HCA (30 μM)  200 minutes                                                   PCE (60 μM)  200 minutes                                                   PCE (100 μM)  600 minutes                                                  TCE (60 μM)  200 minutes                                                 ______________________________________                                    

A graph showing a reduction in the concentration of TCE by thedehalogenase over time is shown in FIG. 17. A graph showing reduction ofHCA by the dehalogenase over time is shown in FIG. 20. TheMichaelis-Menton kinetics for reduction of PBE are shown in FIG. 21.

It was observed that ascorbic acid functions as a co-factor for thedehalogenase. In addition, competition experiments revealed that thedehalogenase preferentially reduces alkenes over alkanes.

Molecular Weight

The size of the dehalogenase, as determined by polyacrylamide gelelectrophoresis, is approximately 293,000 daltons and is believed tocontain two subunits having a molecular weight of approximately 90,000daltons, two subunits having a molecular weight of approximately 39,000daltons, two subunits having a molecular weight of approximately 28,000daltons, and one or more copper and iron molecules.

Oxidation of Reduced Contaminants

Contaminants that have been reduced by the reducing agent as describedabove are oxidized to environmentally safe compounds by the addition ofoxygen. Oxygen is preferably added by bubbling air into the incubationmixture. Exposure of the reduced contaminants to oxygen can also beachieved, for contaminated soil or sediment that has been flooded withwater, by simply removing the water by evaporation or other methodsknown to those skilled in the art.

The remediation method can be carried out in a variety of reactorsincluding batch reactors. Alternatively, the contaminated soil can beremediated in situ without removing the soil from the ground.

Classes of Compounds Reduced

The remediation method can be used to degrade a wide variety ofenvironmental contaminants including, but not limited to, nitroorganiccompounds, halogenated organic compounds, cyano compounds, anisoles, andmetals. It will be understood by those skilled in the art that theremediation method may be used to degrade other contaminants that aresimilarly reduced and oxidized.

a) Nitroorganic Compounds

The remediation method provided herein is particularly useful inreducing nitroorganic compounds contaminating soil, sediment, water oraquifer materials. Nitroorganic compounds are defined herein asnitroaromatics and nitroaliphatics and specifically include munitionssuch as trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine(RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX),N-methyl-N-2,4,6-tetra-nitroaniline (Tetryl), nitrocellulose, andmunition processing wastewater such as the TNT manufacturing by-productknown as "red water". The described remediation method is also usefulfor remediation of nitroorganic pesticide contaminants such as methylparathion and 2-(sec-butyl)-4,6-dinitrophenol, also known as Dinoseb. Inaddition, the remediation method is useful for remediatingnitrobenzenes, benzonitriles such as 4-chlorobenzonitrile, Disperse Blue79, Disperse Red 5, and azo compounds such as azobenzene.

The term "azo compound" as used herein refers to a compound containing a-N═N-- moiety. Disperse Blue 79, Disperse Red 5, and azo-compounds, areindustrial chemicals.

The chemical structures for Disperse Blue 79, Disperse Red 5, parathion,dinoseb, RDX, HMX, and TNT are shown in FIG. 7.

FIG. 8 shows chemical formulas for reduction of several nitroaromaticcompounds by reducing agents in soil to the corresponding anilines in ananaerobic environment. Oxidation of anilines produces catechols whichare degraded to carbon dioxide and the corresponding acetates, as shownin FIG. 10.

b) Other Compounds

The method provided herein is useful for remediation of halogenatedhydrocarbons contaminating soil, sediment, water or aquifer materials.Halogenated hydrocarbons are defined herein as halogenated organiccompounds or solvents such as hexachloroethane (HCA), tetrachloroethene(PCE), tetrabromoethene (PBE), trichloroethene (TCE) andtrichloroethylene, halogenated pesticides and other industrial chemicalssuch as halogenated aromatics and pentachlorophenol (PCP).

The method provided herein is useful for remediating soil, sediment,water or aquifer materials contaminated by cyano compounds such asbenzonitrile, acetonitrile, and other industrial chemicals; anisolessuch as anisole, dyes and pesticides containing methoxy moieties; andmetals such as chromium and arsenic, especially K₂ CrO₄.

The remediation method will be further understood with reference to thefollowing non-limiting examples.

EXAMPLE 1 In Vitro Reduction of para-substituted nitrobenzenes by SixAnaerobic Sediments

A series of para-substituted nitrobenzenes were shown to undergo abioticreduction to the corresponding anilines in several anaerobic sedimentsamples.

Materials

Nitrobenzene, 4-nitrotoluene, 4-nitroanisole, 4-chloronitrobenzene,4-bromonitrobenzene, 4-nitrobenzonitrile, 1,4-dinitrobenzene,4-toluidine, 4-anisidine, 4-chloroaniline, 4-bromoaniline,4-aminoacetophenone, 4-aminobenzonitrile, 4-nitroaniline, catechol,nitrosobenzene (all at least 97% pure; Aldrich Chemical Co., Inc.,Milwaukee, Wis.), 4-nitroacetophenone (97% pure; Pfaltz and Bauer, Inc.,Waterbury, Conn.), aniline (99.5% pure; Fluka Chemical Corp., Hauppauge,N.Y.) were used without further purification. Phenylhydroxylamine, m.p.80-82° C., was synthesized by reducing nitrobenzene with zinc inaccordance with the method of Kamm, O., Org. Syn. 4:57-58 (1924) andrecrystallized from hexane. The identity and purity of these compoundswas confirmed by HPLC. and gas spectrometry-mass spectrometry (GC-MS)using a Finnigan MAT (Finnigan, San Jose, Calif.) automated gaschromatograph/EI mass spectrometer. Acetonitrile (Burdick and Jackson,Musegon, Mich.), sodium hydroxide (Fisher Scientific Co., Pittsburgh,Pa.), nitric acid and hydrochloric acid (J. T. Baker Chemical Co.,Phillipsburg, N.J.) were of high purity.

Sediment

Anaerobic sediments were collected from several different sites. Threeof these sediments were collected near Athens, Georgia from a slowmoving river (the Oconee River), a slow moving high sediment load stream(Beaver Dam), and a small pond (Bar H pond). A fourth sediment wascollected from a bog near Gaston, Ill. (Morgan's Muck). A fifth sedimentwas collected from an uncontaminated aquifer near Quincy, Florida(Florida Aquifer). The sixth sample was collected from a peat bog nearBilthoven, The Netherlands (Loosdrechtsche Plassen). The sediments andassociated water, except the Florida aquifer sample, were collected byscooping up the first 5 to 10 cm of bottom sediment into 1 liter canningjars at a depth of 30 to 50 cm below the water surface. The jars werecapped before being brought to the surface. The samples were sievedthrough a 1 mm sieve in the laboratory to remove debris and stored in aglove box in a nitrogen atmosphere until used. The aquifer sample wascollected using a hollow stem auger with a split spoon sampler in afresh drilled well 15 to 20 meters deep. The sediment was covered withgroundwater, transported to the laboratory and stored in a glove boxunder a nitrogen atmosphere until used.

Sediment Eh readings were taken in the glove box with a Markson 1202combination platinum electrode that was placed in the sediment.(Markson, Houston, Tex.) The electrode was calibrated against a standardferrous-ferric poised standard (Light et al., 1972). If the electrodedid not read to within ±10 mV of Eh=750 mV, the electrode was cleanedwith 10% HCl (v/v) followed by 10% H₂ O₂ (v/v). The measurement wastaken when the Eh reading stabilized (approximately 20 min). Thefraction organic carbon, F_(oc), (w/w) was measured using air driedsediment by coulometric titration using an automated instrument(Dohrman, Santa Clara, Calif.) in accordance with the method of Lee, C.M. and D. L. Macalady, Inter. J. Environ. Anal. Chem. 35:2219-225(1988).

Sediment pH was measured in the glove box with a portable CorningpH/TempMeter 4 equipped with a Ross combination electrode (OrionResearch, Inc., Boston, Mass.). After calibrating the pH meter with pH4.00, 7.00, and 10.00 buffers (Fisher Scientific Co., Pittsburgh, Pa.),the pH was measured by inserting the electrode into a stirred sedimentslurry. Stirring was stopped and the pH was recorded when the readingstabilized (approximately 10 min).

Liquid Chromatography

A Waters 501 chromatographic pump equipped with a Kratos 757 variablewavelength UV-visible detector (Kratos Analytical Instruments, Ramsey,N.J.) and a Rheodyne 7125 injector using a 20 μl sample loop (Rheodyne,Inc., Cotati, Calif.) was used for reverse phase HPLC. Typically, thecolumn was a pH stable Hamilton PRP-1 column, 250 mm long×4.1 mm i.d.,10 μm particle size (Hamilton Co., Reno, Nev.). The analytical columnwas protected with a cartridge guard column containing a PRP-1cartridge, 10 mm×4.6 mm i.d., 10 μm particle size (Alltech Associates,Inc., State College, Pa). Typically the mobile phase wasacetonitrile:water (60:40, v/v) at pH 12 (adjusted with 10 N NaOH), andthe flow rate was 1.0 ml/min at 1500 psi. These conditions were used forthe analysis of both disappearance of the parent compound and productformation. The mobile phase for catechol analyses was acetonitrile:water(20:80, v/v) at pH 1.75 (adjusted with concentrated HNO₃).

Detector wavelength varied with the compound of interest: nitrobenzene,262 nm; 4-nitrotoluene, 272 nm; 4-nitroanisole, 310 nm;4-chloronitrobenzene, 278 nm; 4-bromonitrobenzene, 278 nm;4-nitroacetophenone, 262 nm; 4-nitrobenzonitrile, 272 nm;1,4-dinitrobenzene, 262 nm; aniline, 233 nm; 4-toluidine, 235 nm;4-anisidine, 232 nm; 4-chloroaniline, 240 nm; 4-bromoaniline, 240 nm;4-aminoacetophenone, 230 nm; 4-aminobenzonitrile, 275 nm;4-nitroaniline, 220 nm; catechol, 262 nm; nitrosobenzene, 320 nm;phenylhydroxylamine, 262 nm. Product formation was confirmed by GC/MSand quantitated by HPLC through comparison with known standards. TheGC/MS system used for product formation was a Hewlett-Packard 5890 GCwith a 15 m HP-1 0.2 mm capillary column with 0.33 μm film thicknessconnected to a Hewlett-Packard 5970 mass selective detector (HewlettPackard Co., Palo Alto, Calif.).

Distribution Coefficient

The soil to water ratio was measured by weighing triplicate aliquots ofsediment-water slurry, then evaporating at 110° C. overnight anddetermining the dry weight. The distribution coefficient (K_(d)) of thecompounds were determined in conjunction with most kinetic experiments.This was accomplished by centrifuging spiked sediment-water samplesafter vortexing 1 min and removing a 3.0 ml aliquot of the aqueousphase. The aqueous phase was extracted with 1.0 ml acetonitrile. Theremaining sediment phase was extracted with 1.0 ml acetonitrile andcentrifuged. Both extracts were analyzed via HPLC. Corrections were madefor the water remaining in the sediment phase.

Kinetic Procedures

Kinetic experiments were conducted by a batch method in which 5 ml of astirred sediment-water slurry was pipetted into a series of 10 mlscrew-capped test tubes. The tubes were spiked with 50 μm of a 1.0×10⁻³M solution of the desired nitrobenzene derivative in acetonitrile ormethanol. The test tubes were capped with screw caps fitted with teflonor n-butyl rubber lined septa, removed from the glove box and vortexedfor 10 seconds. All sediment sample manipulations except for the initialsieving were conducted within the glove box. The test tubes wereincubated at the desired temperature (±1° C.) in a water or oil bathwith gentle shaking. Kinetic studies conducted above 100° C. werecarried out in sealed glass ampoules. Periodically a test tube wassacrificed for analysis by adding 1.0 ml acetonitrile then vortexing 1min. The tube was centrifuged (15 minutes at 4000 rpm) and thesupernatant analyzed by HPLC. Typical sample recoveries were in excessof 95% (w/w). To examine the role of pH, the pH of sediments was alteredby the dropwise addition of either 10 N NaOH or concentrated HCl towithin 0.5 pH units of the desired value and allowed to equilibrate 24hours. The pH was measured and adjusted again as necessary. The sampleswere not used until the desired pH had stabilized for 24 hours.

Results

The eight para-substituted nitrobenzene compounds were reduced by thegeneral stepwise reduction mechanisms shown in FIG. 8. Nitrobenzene gaveaniline as the stable product with phenylhydroxylamine andnitrosobenzene as reactive intermediates.

As shown in Table 4, the para-substituted nitrobenzene compounds werereduced when the pH of the sediments was between 5.3 and 10.55. However,at pH 3.85 and 2.50, no reduction of the para-substituted nitrobenzenecompounds could be detected within experimental error. Additionally,sediment flocculation and agglutinization of the solids was observed atthis low pH. At pH 7, K_(d) was 3.4 and Eh was 105 mV. The organiccarbon fraction was 0.0166.

The disappearance rate constant for nitrobenzene correlated best withthe organic carbon content of the solid phase as shown in FIG. 11.

                  TABLE 4                                                         ______________________________________                                        Disappearance rate constants for                                                nitrobenene reduction in Beaver Dam sediment over                             pH range 2.5-10.6                                                                      Sediment/H.sub.2 O                                                   pH ratio k.sub.obs (10-.sup.4 m-.sup.1).sup.a k.sub.corr (10.sup.4                                             m-.sup.1).sup.b                            ______________________________________                                        2.5    0.0288       no observed rxn                                                                            0.25 + 0.025                                   3.85 0.0281 no observed rxn 0.12 + 0.012                                      5.3 0.0433 4.0 ± 0.21 4.6 ± 0.24                                        7.0 0.0305 3.8 ± 0.18 4.2 ± 0.20                                        7.75 0.0314 7.7 ± 0.70 8.5 + 0.77                                          9.15 0.0244 8.6 ± 0.57 9.3 ± 0.62                                       10.55 0.0520 6.6 ± 1.0 7.8 + 1.2                                         ______________________________________                                         .sup.a Observed firstorder disappearance rate constant and standard           deviation.                                                                    .sup.b Calculated maximum rate assuming a 10% analytical method error.   

EXAMPLE 2 In Vitro Reduction of Nitrobenzene Compounds by Four AnaerobicSediments

Nitrobenzene, 4-ethylnitrobenzene, 4-(n-butyl)nitrobenzene and4-(n-octyl)nitrobenzene in anaerobic sediment samples were reduced tothe corresponding anilines.

Chemicals and Sediments

Nitrobenzene (99+%), 4-ethylnitrobenzene (99+%), 4-ethylaniline (99+%),4-(n-butyl)aniline (97%), and 4-(n-octyl)aniline (99%) from AldrichChemical Co., Milwaukee, Wis., and reagent grade aniline from the J. T.Baker Chemical Co., Phillipsburg, N.J., were used without furtherpurification. Formaldehyde (37%) solution, with 12% Methanol, from J. T.Baker Chemical Co.), hydrogen peroxide (30% solution from J. T. BakerChemical Co.), mercuric chloride (Fisher Scientific Co., Pittsburgh,Pa.), and isopropanol (spectrograde, Burdick and Jackson, Musegon,Mich.) were used as chemical sterilants. Sodium azide (Fisher ScientificCo.), toluene (Spectrograde, Burdick and Jackson), and m-cresol (99+%)(Aldrich Chemical Co. Inc., Milwaukee, Wis.) were used as metabolicinhibitors. All solvents used were of high purity grade from Burdick andJackson.

4-(n-Butyl)nitrobenzene and 4-(n-octyl)nitrobenzene were synthesized byslowly adding a 3.0 M methylene chloride solution (2.5 ml) of thecorresponding aniline to a stirred 0.7 M methylene chloride solution (45ml) of 3-chloroperoxybenzoic acid (tech., 80-85%, from Aldrich ChemicalCo. Inc.). After refluxing for one hour and cooling, the mixture waswashed with 1.0 N NaOH, then 0.1 N HCl, then water, and then the organiclayer was dried over sodium sulfate. The methylene chloride wasevaporated under a nitrogen stream, leaving a yellow-orange liquid. NMRand IR spectra showed no remaining amino compound, and both nitrocompounds gave a single peak via liquid chromatography. Yields for4-(n-butyl)- and 4-(n-octyl)nitrobenzenes were 77 and 62%, respectively.

Thioglycollate indicator was used to check anaerobic sterility. Thethioglycollate indicator agar was made by bringing thioglycollate medium(14.5 g, Difco Laboratories, Detroit, Mich.), BACTO-AGAR™ (5 g, DifcoLaboratories), and 0.1% resazurin indicator (5 ml, resazurin from SigmaChemical Co., St. Louis, Mo.) to a boil in 500 ml of water, withsubsequent cooling. The resazurin indicator turns the nutrient mediumred if conditions become aerobic. Tryptone Glucose Extract Agar (DifcoLaboratories) was used to assay for aerobic sterility.

Sediments

Anaerobic sediment samples from four bodies of water near Athens, Ga.were used. Three of these (known as Hickory Hill, Memorial Park, andBar-H) were samples from lakes; the fourth (Beaver Dam) was from astagnant, slow moving stream. Sediments were collected by scooping upsamples in 1 quart canning jars at a depth of 1-2 feet below the watersurface. The jars were capped, brought back to the laboratory, passedthorough a 1 mm sieve, and stored in a glove box under a nitrogenatmosphere until used. Sediments were used within two weeks ofcollection.

Liquid Chromatography

A Tracor 950 chromatographic pump equipped with a Tracor 970A variablewavelength detector (Tracor Instruments, Austin, Tex.) and a Rheodyne7120 injector with a 50 μl sample loop (Rheodyne, Inc., Cotati, Calif.)was used for liquid chromatography. The column was a MicromeriticsMicrosil™ C-18, 25 cm length×4.6 mm I.D., 5 μm particle size(Micromeritics Instrument Co., Norcross, Ga.). A guard column (2 mmI.D.×7 cm length) was filled first with a small amount of Microsil™ 5 μmpacking and then with Whatman 30-38 μm pellicular C-18 guard columnpacking (Whatman SA, France). For 4-(n-octyl)nitrobenzene, the solventwas 70:15:15 acetonitrile:tetrahydrofuran:water. The elution time was4.3 minutes behind the solvent front (flow rate 1.5 ml/min). For allother compounds, the solvent was 70:30 acetonitrile:water. Nitrobenzene,4-ethylnitrobenzene, and 4-(n-butyl)nitrobenzene eluted 2.3, 3.0 and 5.5minutes behind the solvent front, respectively. In each case, thecorresponding aniline eluted slightly earlier than its nitro analog. Thewavelength used was 280 nm.

Kinetic Experiments

The pH was determined before each experiment using an Orion 91.62 probe(Orion Research, Inc, Boston, Mass.) by gently agitating the probe inthe solid phase at the bottom of the jar. In all four sediment samples,the pH was maintained between 6.8 and 7.1. Sediment Eh readings werealso taken before each experiment by placing a Markson 1202 combinationplatinum electrode (Markson, Houston, Tex.) in the sediment jarovernight to equilibrate before taking readings. The Eh generally readbetween -170 and -230 mv.

Aliquots (5 ml) of the desired sediment, drawn while stirring thesediment with a glass rod in the glove box, were transferred to a seriesof 15 ml screw cap test tubes. The tubes were capped with Hungate (opentop) screw caps (fitted with teflon lined septa) and brought outside ofthe glove box. The tubes were spiked with 5 μl of a tetrahydrofuranstandard (10⁻² M) of the desired nitroaromatic compound. The tubes werebriefly vortexed. At selected time intervals, one tube from the serieswas sacrificed by quenching with acetonitrile (2 ml) and vortexing for30 seconds. The remaining sample tubes were inverted four times at thispoint. For nitrobenzene, 4-ethylnitrobenzene, and4-(n-butyl)nitrobenzene, the tubes were then centrifuged (tabletopcentrifuge, 2500 rpm for 20 minutes) and the supernatant (5 ml) wasfiltered through a 925 mm Millipore™ filter (Millipore Corp., Bedford,Mass.). The sample was then ready for analysis. For4-(n-octyl)nitrobenzene, hexane (4 ml) was added to the quenched sample,and it was vortexed again for 1 minute. An aliquot (3 ml) of the hexanelayer was removed, evaporated under a nitrogen stream, and redissolvedinto acetonitrile (5 ml). The sample was then ready for analysis.

For runs with added chemical sterilant or inhibitor, the appropriateamount of chemical was added to the sediment 16 hours before spikingwith nitrobenzene. For kinetic runs without the sand and silt fractions,the sediment was allowed to settle for 1.5 hours under nitrogen afterstirring before aliquots were drawn. For kinetic runs with sedimentassociated water only, sediment was centrifuged as above, and theresulting supernatant was used for kinetic runs. Recoveries of thenitroaromatic compounds 4 hours after spiking from twice-autoclavedsediment (220° C., 20 psi×20 m) were 84±3% for nitrobenzene,4-ethylnitrobenzene, and 4-(n-butyl)nitrobenzene, and 100±5% for4-(n-octyl)nitrobenzene.

Sediment/Water Ratios

Triplicate aliquots (5 ml) of sediment were transferred to tared testtubes. After reweighing, the samples were centrifuged as above, thesupernatant removed, and the pellet dried in an oven overnight at 95° C.After cooling, the dried samples were weighed again. Ratios werecalculated by dividing the dry weight of the sediment by the weight ofthe aqueous phase.

Distribution Coefficients

Distribution coefficients (K_(d)) for the four nitroaromatics weredetermined at 3 minutes, 1 hour, 3 hours, and 4 hours after spiking intwice autoclaved sediment (as above). Samples were periodicallyinverted. For nitrobenzene and 4-ethylnitrobenzene, the spiked samplewas centrifuged (as above) after the desired incubation time, andsupernatant (3.5 ml) was removed and filtered as described above. To theremaining supernatant and pellet, acetonitrile (2 ml) was added, and thesample was vortexed for 30 seconds. The sample was then centrifugedagain, and the supernatant (2-2.5 ml) was filtered. After correcting forthe supernatant contribution to the pellet extract, the K_(d) (6.25 μgcompound/g dry solid)/(μg compound/g supernatant) was calculated usingthe sediment/water ratios calculated for the experiment. Recoveries werequantitative for both compounds. For 4-(n-octyl)nitrobenzene, sedimentdiluted 10-fold with supernatant from another sample jar (obtained bycentrifugation) was used. After the desired incubation time, the samplewas centrifuged, and 4 ml of supernatant was removed and centrifugedagain. A portion of this second supernatant (3 ml) was removed andextracted with hexane (2 ml). An aliquot of the hexane layer (1.5 ml)was then evaporated under a nitrogen stream, and the residue redissolvedin acetonitrile (1 ml) before analysis. To the pellet and remainingsupernatant, acetonitrile (2 ml) and hexane (4 ml) were successivelyadded, vortexing for 30 seconds and 1 minute afterwards, respectively.An aliquot of the hexane layer (3 ml) was then evaporated and theresidue was redissolved in acetonitrile (5 ml) for analysis. The K_(d) Swere calculated after correcting for the sample handling steps. Therecovery was 86±4% for 4-(n-octyl)nitrobenzene.

Diluted sediment was also used to determine the K_(d) for(n-butyl)nitrobenzene. The K_(d) was determined in a similar manner asfor 4-(n-octyl)nitrobenzene, except that acetonitrile alone (4 ml) wassufficient to extract the pellet, and after a second centrifugation, analiquot of the extract (3 ml) was filtered and analyzed directly.Recoveries were only 60±6% for 4-(n-butyl)nitrobenzene, but this methodwas found to give the best recoveries of the extraction methodsattempted. K_(d) were found to remain nearly constant with time for allof the above compounds.

Aerobic and Anaerobic Sterility Tests

Aerobic sterility was determined by applying 0.1 ml of treated sedimentto sterilized, hydrated tryptone glucose extract agar spread on a Petridish and watching for growth within 5 days. Anaerobic sterility wasdetermined by stabbing treated sediment into 5 ml plugs of sterilizedthioglycollate indicator agar under nitrogen in 15 ml test tubes.Sterility was indicated by the retention of the pink oxygenated band atthe top of the plug, the absence of gas formation, and no visible growthin the anaerobic portion of the agar over a five day period.

Polarography

The reduction potential of the four nitroaromatics was measured inaqueous solution (10 mM) at pH 6.8 (0.1 M phosphate) using square wavepolarography. The instrument was an EG & F Princeton Applied ResearchCorp. Digital Polarograph 82 (EG & F Instruments, Ltd., United Kingdom)used in the HMDE mode, with a medium drop size. The purge time was 4minutes (helium). The scan range was -100 to -600 mV (relative toAg/AgCl); pulse size, -20 mV; step size, -5 mV; drop settle delay, 400ms; sweep delay, 0 m; preintegration time, 2000 μs; integration time12,562 s; and current gain 256.

Results

As shown in Table 5, nitrobenzene was readily degraded in all samples ofnitroaromatic anaerobic sediment gathered from the four different waterbodies. The half-lives for the reduction of nitrobenzenes in anaerobicsediment samples are on the order of a few hours as shown in Table 6. Agraph showing the reduction of nitrobenzene to aniline over a two hourperiod of time is shown in FIG. 9.

Nitrobenzene in sediment which had been autoclaved exhibited a reducedrate of reduction to aniline. Chemical sterilization with formaldehydehad no effect on the rate of reduction to aniline. Therefore, thereducing component is heat labile but not labile to formaldehyde whenbound to sediment.

                                      TABLE 5                                     __________________________________________________________________________    Reduction of nitrobenzene in four anaerobic sediments                         Sediment                                                                            % OM.sup.a                                                                         Eh(mv)                                                                             p      K.sub.d                                                                            t.sub.1/2 (min)                                                                    r.sup.2                                      __________________________________________________________________________    Beaver Dam                                                                          5.6 ± 0.3                                                                       -170 0.112 ± 0.016                                                                     3.9 ± 0.8                                                                       142 ± 51                                                                        0.980                                          Bar-H 2.2 ± 1.0 -170 0.060 ± 0.006 2.6 ± 0.5 21 0.999                Hickory 1.8 ± 1.0 -230 0.052 ± 0.002 2.6 ± 0.4 142 0.999                                             Hill                                          Memorial 4.3 ± 0.2 -250 0.038 ± 0.003 9.0 ± 1.0 120 0.991                                            Park                                        __________________________________________________________________________     .sup.a Deterinined by heating dry sediment at 425° C. for 20 hours     corrected for water of adhesion (obtained by heating dry sediment at          70° C. for 3 days)                                                

                  TABLE 6                                                         ______________________________________                                        Pseudo-first-order disappearance rate constants for reduction of                nitrobenzene and three 4-substituted nitrobenzenes                                 Compound       t.sub.1/2 (min)                                         ______________________________________                                        nitrobenzene      53                                                            4-ethylnitrobenzene 74                                                        4-(n-butyl) nitrobenzene 120                                                  4-(n-octyl) nitrobenzene 1140                                               ______________________________________                                    

EXAMPLE 3 Kinetics of Reduction of 15 Halogenated Hydrocarbons in FourAnoxic Sediment Samples

Sediment-water slurries were collected from the ponds known as VechtenPond, Bilthoven; Breukelveen, and Loosdrechtse Plassen and theslow-moving stream known as Dommel, all in The Netherlands. Samples werecollected by scooping the first 5-10 cm of bottom sediment into glassjars. The jars were completely filled with sediment and water and cappedunder the water surface. Sediment samples were stored at 20° C. untilused for experiments. Prior to use, the samples were sieved through a 1mm wire sieve to remove debris. The sediment to water ratio (g/g) ofsamples was determined by placing 10 ml aliquots of the thoroughly mixedsediment-water sample in weighed, open 50 ml jars. Consequently thewater was evaporated during one day at 80° C. and the jars werereweighed. Each determination was repeated five times.

Kinetics experiments were performed using a batch method in whichsediment aliquots were distributed into a series of test tubes andspiked with a known concentration of a halogenated hydrocarbon under anitrogen atmosphere. All halogenated hydrocarbons were purchased fromAldrich Chemical Co., Milwaukee, Wis. A tube was sacrificed for analysisof the concentration of chemical in the sample at specific timeintervals during incubation. For each experiment, 10 ml aliquots ofsediment were placed in 20 ml test tubes. A stock solution of eachhalogenated compound was made in methanol (Burdick and Jackson, Musegon,Mich.) such that a 10 μl addition of chemical into 10 ml sediment gavethe desired initial experimental concentration. Sample tubes were spikedwith 10 μl halogenated hydrocarbon. After vortexing, the tubes wereincubated at 22° C. and were periodically mixed. At specific timeintervals, 2 ml of acetonitrile (Burdick and Jackson) was added to thetubes to quench the reaction. Tubes were vortexed for 1 minute, thesediments extracted with 4 ml cyclohexane (Burdick and Jackson) andvortexed again for 2 minutes. The cyclohexane layer was recovered fromthe tubes after centrifugation at 3500-4000 rpm for fifteen minutes,placed in a clean tube and stored at -30° C.

Cyclohexane extracts were analyzed using a Carlo Erba (Milan, Italy)4160 gas chromatograph equipped with an electron-capture detector and aHewlett Packard (Avondale, Pa.) 3392 integrator. The column used was afused silica open tubular column with CP-sil-5CB as the stationary phase(25 m×0.32 mm). The relative concentrations of the compounds werecalculated by comparing peak areas of samples at given times against thepeak area of the zero time sample.

A Metrohm (Herisau, Switzerland) pH meter was used for both the pH andEh measurements. Eh values were measured using a platinum Ag:AgClreference electrode. All Eh values are reported versus SHE. All pH andEh measurements were performed under a nitrogen atmosphere.

GC-MS analyses were performed using a Hewlett Packard model 5890A gaschromatograph interfaced with a Finnigan (San Jose, Calif.) 4500quadrupole mass spectrometer. The column was a fused silica capillarycolumn with CP Sil-5 as the stationary phase.

The NMR spectra were recorded at 200 MHz on a Bruker (Karlsruhe,Germany) AC 200 NMR spectrometer interfaced with an ASPECT 3000computer. The spectra were recorded using CDCl₃ (deuterated chloroform)as the solvent with TMS (tetra-methyl silane) as the internal standard.

The results of the kinetics studies for 15 halogenated hydrocarbons areshown in Table 7. The Eh and pH did not change appreciably during theexperiment. Below an Eh value of -50 mV, the disappearance rate constantwas essentially independent of the Eh. The rate of disappearance of allthe compounds that reacted was first-order through at least twohalf-lives. The halogenated hydrocarbons exhibited a wide range ofreactivity with the shortest half-life of about ten minutes fortetraiodoethene to no detectable reaction after 90 days of incubationfor perflurodecaline, perfluorohexane and DDT. A calculated maximum rateconstant and minimum half-life can be arrived at by assuming a 10%experimental error in the analysis of the compound after 90 daysreaction time. All of the halogenated hydrocarbons were stable tohydrolysis under the redox reactions employed in this example.

                  TABLE 7                                                         ______________________________________                                        Kinetic data and reaction parameters                                            for reduction of halogenated hydrocarbons in                                  sediment samples under anaerobic conditions                                                   Initial    Rate    Half                                        Conc. Constant life                                                          Compound (mol/l) k (min-1) (hour)                                           ______________________________________                                        Tetraiodoethene.sup.1                                                                       9.60E-06   9.22E-02  0.1                                          Hexachloroethane.sup.2 7.32E-08 2.47E-02 0.5                                  Hexachloroethane.sup.3 7.32E-08 7.62E-03 1.5                                  1,2-Dibromo-1,2- 1.21E-06 5.44E-03 2.1                                        dichloroethane.sup.1a                                                         1,2-Dibromo-1,2- 1.21E-06 4.54E-03 2.5                                        dichloroethane.sup.1a                                                         Hexachloroethane.sup.1 7.32E-08 2.33E-03 5.0                                  Hexachloroethane.sup.4 7.32E-08 1.22E-03 9.5                                  Carbontetrachloride.sup.2 1.6E-06 1.16E-03 10.0                               2,3-Dibromobutane.sup.1a 1.19E-06 1.10E-03 10.5                               2,3-Dibromobutane.sup.1a 1.19E-07 9.15E-04 12.6                               1,2-Dibromoethane.sup.1 9.28E-07 7.13E-04 16.2                                Hexachloro- 5.70E-07 1.51E-04 76.5                                            cyclohexane.sup.1                                                             Tetrachloroethene.sup.1 9.79E-08 4.05E-05 285.5                               Iodobenzene.sup.1 2.2E-06 2.55E-05 453.0                                      Hexachloro- 5.09E-07 2.36E-05 489.4                                           cyclohexane.sup.1                                                             DDT.sup.1 1.44E-06 NR --                                                      Perfluorodecaline.sup.1 1.44E-06 NR --                                        Perfluorohexane.sup.2 2.01E-06 NR --                                        ______________________________________                                         .sup.1 Sediment obtained from Vechten Pond: pH 7.2, Eh139 mV, Organic         Carbon 6.0%.                                                                  .sup.2 Sediment obtained from Breukelveen: pH 7.6, Eh145 mV, Organic          Carbon 29%.                                                                   .sup.3 Sediment obtained from Dommel: pH 7.5, Eh155 mV, Organic Carbon        0.53%                                                                         .sup.4 Sediment obtained from Loosdrechtse Plassen: pH 7.7, Eh128 mV,         Organic Carbon 32.6%                                                          NR = no reactivity observed durinmg incubation; * = Diastereomer         

EXAMPLE 4 In vitro Remediation of Hexachloroethane in 18 Sediment orAquifer Samples

The sorption-corrected rate constants for the reduction of thehalogenated hydrocarbon hexachloroethane (Aldrich Chemical Co.,Milwaukee, Wis.) by reducing agents present in 18 different sediment,soil and aquifer samples were correlated with organic carbon content.

Rate constants for reduction of hexachloroethane were determinedgenerally as described in Example 3.

Organic carbon content was determined by the method of Lee and Macalady,Inter. J. Environ. Anal. Chem. 35:219-225 (1988).

Correlation of Rate Constants with Organic Carbon Content

Disappearance rate constants for the reduction of hexachloroethane byreducing agents in 18 different sediment, soil and aquifer samples arelisted in Table 8, in the order of decreasing rate constants (k_(obs)).The data in Table 8 suggest a relationship between the rate constantsand organic carbon content of the samples.

                  TABLE 8                                                         ______________________________________                                        Measured (k.sub.obs) and sorption corrected (k.sub.corr)                        disappearance rate constants for hexachloroethane                             in selected sediments and aquifer samples.                                                         Sediment                                               SEDIMENT Fraction  conc.     log K.sub.obs *                                                                       log k.sub.corr.sup.#                       SOURCE OC (g.g.sup.-1) min.sup.-1 min.sup.-1                                ______________________________________                                        BarH     0.022     0.08      -1.301  -1.081                                     BarH 0.022 0.09 -1.455 -0.248                                                 HickoryH 0.018 0.11 -1.585 -0.519                                             Breukelveen 0.29 0.045 -1.607 0.407                                           BeaverD 0.056 0.2 -1.699 -0.570                                               MemorP 0.043 0.055 -1.721 -0.841                                              BarH 0.022 0.075 -1.721 -0.586                                                Loosdr. 0.33 0.050 -2.118 -0.018                                              Plassen                                                                       Vechten 0.06 0.087 -2.632 -1.019                                              Pond                                                                          EPA-B1 0.009 0.1 -2.745 -1.847                                                Dommel 0.0053 0.469 -2.915 -1.613                                             EPA-13 0.03 0.1 -3.089 -1.708                                                 EPA-11 0.015 0.1 -3.104 -2.007                                                EPA-6 0.0072 0.1 -3.350 -2.535                                                Lula, aq 0.000065 0.161 -4.267 -4.233                                         Blythville, 0.00012 0.142 -4.359 -4.306                                       aq                                                                            Blythville, 0.00012 0.613 -4.361 -4.167                                       aq                                                                            Lula, aq 0.000065 0.689 -4.521 -4.393                                       ______________________________________                                         *observed rate constants                                                      #sorption corrected rate constants: k.sub.corr  = k.sub.obs  * (1 + ρ     * k.sub.d)                                                                    aq = aquifer                                                             

Sorption Corrected Rate Constants

The sorption corrected rate constant (k_(corr)) in which k_(obs) iscorrected for the fraction of the compound sorbed is given by thefollowing equation, in which P denotes the sediment concentration (g.g⁻¹):

    k.sub.corr =k.sub.obs *(1+P*K.sub.d)                       (4)

The k_(corr) values are included in Table 8 and were calculated using alog K_(ow) (octanol-water partition coefficient) value of 4.61. Linearregression analyses showed that this correction for sorption increasesthe correlation between organic carbon content and the disappearancerate constant.

Although there are other factors that contribute to the reduction ofhexachlorethane, organic carbon content accounts for about 91% of thevariance of the data.

EXAMPLE 5 Nitroreduction of 2,4,6-Trinitrotoluene (TNT) with CrudeEnzyme Extract

The effects of initial TNT concentration on the reaction order inanaerobic sediment samples, the reaction of TNT with crude proteinsextracted from high organic carbon content sediments, and the effect ofiron powder on TNT degradation in flooded soil were analyzed.

To monitor the disappearance of TNT and formation of its products inanaerobic sediment samples, TNT and its reduced products were extractedfrom water/sediment samples with acetonitrile (Burdick and Jackson,Musegon, Mich.) and analyzed by HPLC equipped with a UV detector asfollows. The solvent was 30% water (pH 10) and 70% acetonitrile at aflow rate of 0.7 ml/min. Absorbance was detected at a wavelength of 238nm. A representative HPLC chromatogram for TNT and its reduced productsis shown in FIG. 12. The dinitro and monoamino toluene isomers (Peak 2)eluted at same retention time, however, these isomers can be separatedand identified by GC/MS. The two diamino, mononitrotoluene isomers (Peak3) also co-eluted. Triaminotoluene (Peak 4) eluted with the solventfront. TNT (Peak 1) and standards for identification of its reducedproducts were obtained from the U.S. Army Research, Development, andEngineering Center, Picatinny Arsenal, N.J.

The dependence of initial concentration of TNT on reaction order wasinvestigated. The initial concentrations of TNT used were 0.25 ppm, 2.5ppm and 125 ppm. The saturated concentration of TNT is about 125 ppm inwater.

Sediments were collected from a stagnant, slow moving stream nearAthens, Ga. These sediments were passed through a 1 mm sieve. Aliquotsof 5 ml of sediment slurries were transferred to a series of 15-ml screwcap test tubes and kept under anaerobic condition. The sediment/waterratio was 0.12, pH was 6.5 and Eh value was -368 mv (vs. Ag/AgCl). Acertain amount of concentrated TNT solutions were spiked into sedimentsamples to achieve three concentrations of TNT. At given time intervals,one tube of sample from the series was extracted by adding 1 ml ofacetonitrile, vortex-mixing for 1 min and centrifuging. The supernatantwas transfer centrifuged again and analyzed. FIG. 13 shows percentage ofTNT remaining in the water/sediment sample as a function of time forthree different concentrations of TNT. The rate of change of TNTconcentration is a constant when the concentration of TNT is 125 ppm orgreater. Beyond this concentration, the reaction proceeds at a rateindependent of both concentrations of TNT and sediment. Therefore, athigh TNT concentration, the reaction rate is zero order. When theconcentration of TNT was decreased to 2.5 ppm, the concentration of TNTdecreased exponentially with time. Plotting ln(c/c0) against time byusing least-square regression method, a straight line was obtained withthe value of r² equal to 0.99. This reaction of TNT is a first orderreaction and half-life is 90 min. With 0.25 ppm TNT, the reduction ratewas fast. After a certain elapsed time, the concentration change sloweddown. A plot of 1/c as a function of time and regression analysis showedthat the TNT reduction in water/sediment system appeared to follow asecond order reaction. This result assumed that both TNT and sedimentactivity have the same initial stoichiometric concentration.Consequently, the results indicated that the reaction order of TNT inthe water/sediment system depends on TNT initial concentration.

The reducing activity of a protein extract isolated from a high organiccarbon sediment was analyzed after introduction into either an aqueoussolution or a low organic carbon content system such as aquifermaterial. One ppm TNT solution was mixed with 0.5 g/ml aquifer sample asthe control. One ppm TNT solution was mixed with 15 μg/ml of theTNT-reducing protein extract isolated from fraction 18 of FIG. 2 asdescribed above and incubated for 24 hours. The purpose of incubationwas to let the substrate, TNT, associate with the denatured subunits torecover the original active form of the enzyme. This procedure wasfollowed by the "Microbial Metabolism of Aromatic Nitriles" studydescribed by David B. Harper, Biochem. J. 167:685-692 (1977). After theincubation period, the incubated sample was spiked with concentrated TNTsolution so that the final TNT concentration was 1 ppm.

To determine whether the protein extract would bind to aquifer materialand retain activity in water, 15 μg/ml of protein extract was incubatedwith 0.5 g/ml aquifer sample for 24 hours before the TNT solution wasadded. The aquifer material had been collected from Columbus Air ForceBase in Georgia. The organic carbon content in this aquifer was 0.027%.

FIG. 14 shows the kinetics of TNT degradation in the three systemsdescribed above. When Ln(c/c0) was plotted as a function of time, theregression analysis showed that the reaction of TNT follows first-orderkinetics in all three systems. A comparison of these three resultsindicates that the protein extract bound to sediment enhances the rateof disappearance of TNT. Half-life of TNT in the combined aquifermaterial and protein extract system is smaller than that in proteinextract system alone.

The effects of the addition of iron to the system was analyzed. 1.6 ppmTNT in distilled water was combined with 1% (w/v) iron powder underanaerobic conditions. As shown in FIG. 15, a plot of ln(c/c0) as afunction of time using the regression method gave a straight lineindicating that the TNT reduction is a first order process with ahalf-life of 1.14 days. The TNT reduction rate was increased by shakingthe reactor to enhance the mass transfer. It should be noted that TNTreduced products were not observed in this study, most likely becausethe reaction rate of TNT reduced products on the metal surface wasextremely fast.

A bench scale experiment was designed to investigate the redoxdegradation of TNT in contaminated soils. TNT contaminated soils weresampled from Alabama Army Ammunition Plant near Childersburg, Ala. 100 gof contaminated soil was flooded by water to a volume of 900 ml in aquart jar. At given time intervals, aliquots of 1-1.5 ml were taken fromthe reaction mixture and centrifuged. The supernatant was decanted andthe sediment was extracted with acetonitrile. Both supernatant andsediment extracts were analyzed. FIG. 16 shows TNT concentrationremaining in supernatant and solid phase as a function of time. Theinitial concentration of TNT in contaminated soil was 6000 ppm based ondry soil. In this study, the concentration of TNT remaining in the solidphase based on wet soil was determined. The concentration of TNT in bothaqueous and solid phases were almost constant during 48 day period.Also, Eh measured in the glove box was very positive. At day 48, 9 gramsof iron powder was added to the reaction system. Although theconcentration of TNT in solid phase did not change significantly, boththe concentration of TNT in supernatant and the Eh decreased with time.When the reaction jar was inverted and shaken vigorously, both TNT inthe supernatant and solid phases decreased dramatically. Therefore masstransfer enhances the degradation of TNT.

Modifications and variations of the contaminant remediation method andreducing agent composition will be obvious to those skilled in the artfrom the foregoing description. Such modifications and variations areintended to come within the scope of the appended claims.

We claim:
 1. A reducing agent for chemical reduction of an environmental contaminant comprising at least one isolated plant enzyme, the enzyme having a molecular weight greater than 5000 daltons and less than 650,000 daltons, wherein the enzyme is an enzyme of a common aquatic weed and has chemical reducing activity for remediation of an environmental contaminant selected from the group consisting of nitroorganics, halogenated hydrocarbons, cyano compounds, and anisoles, wherein the enzyme is not ribulose 1,5-biphosphate carboxylase/oxygenase.
 2. The reducing agent of claim 1 wherein the enzyme is obtained from a sediment sample extract.
 3. The reducing agent of claim 1 wherein the enzyme is an enzyme of the hornwort.
 4. The reducing agent of claim 1 wherein the enzyme has subunits having a molecular weight selected from the group consisting of 19,000, 37,000, 66,000 and 150,000, and wherein one or more subunits combine to form an enzyme.
 5. The reducing agent of claim 1 wherein the enzyme has a molecular weight of approximately 316,000 daltons and nitroorganic compound reducing activity.
 6. The reducing agent of claim 5 wherein the enzyme has a molecular weight of approximately 316,000 daltons and 2,4,6, -trinitrotoluene (TNT) reducing activity.
 7. The reducing agent of claim 1 wherein the enzyme has a molecular weight of approximately 600,000 daltons and nitroorganic compound reducing activity.
 8. The reducing agent of claim 1 wherein the enzyme has a molecular weight of approximately 293,000 daltons and halogenated hydrocarbon reducing activity.
 9. The reducing agent of claim 8 wherein the enzyme has a molecular weight of approximately 293,000 daltons and the ability to chemically reduce a halogenated hydrocarbon selected from the group consisting of hexachloroethane, tetrachloroethene, tetrabromoethene, and trichloroethene.
 10. The reducing agent of claim 1 in combination with a contaminated environmental material selected from the group consisting of soil, sediment, water and aquifer material. 